Prakash et al. Organic and Medicinal Chemistry Letters 2011, 1:1 http://www.orgmedchemlett.

com/content/1/1/1

ORIGINAL

Open Access

A facile iodine(III)-mediated synthesis of 3-(3-aryl1-phenyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-a] pyridines via oxidation of 2-((3-aryl-1-phenyl-1Hpyrazol-4-yl)methylene)-1-(pyridin-2-yl)hydrazines and their antimicrobial evaluations
Om Prakash1*, Khalid Hussain2, Deepak K Aneja2, Chetan Sharma3 and Kamal R Aneja3
Abstract
Background: Fused heterocyclic 1,2,4-triazoles have acquired much importance because of their interesting biological properties. Although a number of methods have been reported in the literature which includes oxidation with phosphorus oxychloride, lead tetraacetate, bromine, etc., hypervalent iodine reagents have emerged as reagents of choice for various synthetically useful transformations due to their low toxicity, ready availability and ease of handling. Results: A series of new 3-(3-aryl-1-phenyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-a]pyridines 4 has been conveniently synthesized by oxidative cyclization of 2-(3-aryl-1-phenyl-1H-pyrazol-4-yl)methylene)-1-(pyridin-2-yl)hydrazines 3 promoted with iodobenzene diacetate under mild conditions (up to 90% isolated yields). All the new compounds were tested in vitro for their antimicrobial activity. Conclusions: Iodine(III)-mediated oxidative approach has offered an easy access to new 3-(3-aryl-1-phenyl-1Hpyrazol-4-yl)-[1,2,4]triazolo[4,3-a]pyridines 4. The antibacterial and antifungal activities of newly synthesized compounds have proved them potent antimicrobial agents. Keywords: hypervalent iodine, antimicrobial activity, triazoles, pyrazole

Background Fused heterocyclic 1,2,4-triazoles have acquired much importance because of their CNS depressant [1], antiallergy [2], antimicrobial [3] and anti-inflammatory [4] properties. Most methods for the preparation of fused 1,2,4-triazole derivatives are based on the oxidation of heterocyclic hydrazones or hydrazides with phosphorus oxychloride [5], lead tetraacetate [5,6], bromine [6,7], etc., which are associated with toxic properties. Therefore, alternative approach avoiding these reagents is always preferred. Organohypervalent iodine reagents have emerged as reagents of choice for various synthetically useful
* Correspondence: dromprakash50@rediffmail.com 1 Institute of Pharmaceutical Sciences, Kurukshetra University, Kurukshetra 136119, Haryana, India Full list of author information is available at the end of the article

transformations due to their low toxicity, ready availability and ease of handling [8-17]. We have recently reported the usefulness of iodobenzene diacetate (IBD) to effect oxidative cyclization of benzalhydrazones to 1,2,4-triazoles [18-22]. Pyrazoles form an integral part of many natural products of therapeutic importance and possess potentially reactive sites for a variety of chemical reactions to generate molecular diversity. (S)-3-Pyrazolylalanine [23], lonazolac [24], difenamizole [25], mepirizole [26], metamizol [27] and 4,5-dihydro-3-phenyl-6H-pyrrolo[1,2-b] pyrazole are some of the biologically active compounds endowed with antimicrobial [28], hypoglycaemic [29] and non-nucleoside HIV-1 reverse transcriptase inhibitor properties [30]. Our ongoing programme on the development of hypervalent iodine-mediated methodologies in heterocyclic

2011 Prakash et al; licensee Springer This is an Open Access article distributed under the terms of the Creative Commons Attribution License

Prakash et al. Organic and Medicinal Chemistry Letters 2011, 1:1 http://www.orgmedchemlett.com/content/1/1/1

Page 2 of 9

synthesis coupled with the significant biological importance of fused 1,2,4-triazole derivatives and pyrazole derivatives, prompted us to undertake the synthesis of hitherto unknown fused 1,2,4-triazolopyridines. We report in this study on the synthesis of fused 3-(3-aryl-1phenyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-a]pyridines 4 by the oxidation of 2-((3-aryl)-1-phenyl-1H-pyrazol-4-yl) methylene)-1-(pyridin-2-yl)hydrazines 3 using IBD in dichloromethane with an expectation to find new and more potent antibacterial and antifungal agents.

Results and discussion

Chemistry

First, we synthesized a series of 2-((3-aryl-1-phenyl-1Hpyrazol-4-yl)methylene)-1-(pyridin-2-yl)hydrazines 3 needed for their oxidative cyclization. These substrates were easily accessible in high yields (88-96%) and purity from the reaction of 2-pyridyllhydrazine 1 and 3-aryl-1phenyl-1H-pyrazole-4-carbaldehydes 2 in ethanol (Scheme 1) [31]. Then, the reaction of 2-((1,3-diphenyl1H-pyrazol-4-yl)methylene)-1-(pyridin-2-yl)hydrazine (3a) (see Additional file 1) was carried out with 1.1 equivalents of IBD in dichloromethane by stirring at room temperature overnight. The usual work-up of the reaction afforded the expected product, 3-(1,3-diphenyl1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-a]pyridine (4a) (see Additional file 2) in 90% yield (Scheme 1). To study the scope of reaction, we carried out oxidation of a wide range of substituted 2-((3-aryl-1-phenyl-1H-pyrazol-4-yl) methylene)-1-(pyridin-2-yl)hydrazines (3b-g) (see Additional files 3, 4, 5, 6, 7 and 8) under similar conditions. It was observed that IBD-mediated oxidative approach worked nicely to give the desired products 4b-g (see

Additional files 9, 10, 11, 12, 13 and 14) in all cases in 82-90% yields. The structures of all the compounds 3 and 4 were confirmed by their spectral (IR, 1 H NMR, Mass) and elemental analytical data. For example, the IR spectrum of the compound 3a exhibited characteristic absorption band at 3190 cm-1 due to NH functional group. The 1H NMR spectrum of the product 3a showed two singlets due to C(5)-H of pyrazole ring and N=CH at 8.93 and 8.17, respectively, and also a broad singlet due to NH at 10.66 which disappeared on the addition of D 2O. Other protons appeared as multiplet in the aromatic regions. Mass spectrum of the compound 3a exhibited molecular ion peak at m/z 340.06 [M + 1]+. The characterization of products 4 was based upon a careful comparison of their IR and 1 H NMR spectra with those of 3. IR spectra of 4 were found to be transparent in the region of NH stretch, thus confirming the oxidation of 3 into 4. An important characteristic feature in the 1H NMR spectra of 4 was the disappearance of the singlet due to N=CH around 8.12-8.93, which was present in the spectra of 3. The plausible mechanism for the oxidation of 3 to 4 is analogous to our earlier reports [18,19] and given in Scheme 2.
Pharmacology In vitro antibacterial activity

All the synthesized compounds, 3a-g and 4a-g were evaluated in vitro for their antibacterial activity against two Gram-positive bacterial strains, Staphylococcus aureus and Bacillus subtilis and two Gram-negative bacteria namely, Escherichia coli and Pseudomonas aeruginosa, and their activity was compared to a well-known commercial antibiotic, ciprofloxacin. All the compounds possessed variable antibacterial activity against Grampositive bacteria, S. aureus and B. subtilis. Results of antibacterial evaluation are summarized in Table 1 and Figure 1. Compounds 3a-g and 4a-g showed zone of inhibition ranging between 12.8 and 24.6 mm. On the basis of zone of the inhibition produced against the test bacteria, compound 3d was found to be the most effective against S. aureus and B. subtilis showing the maximum zone of inhibition of 18.6 and 19.3 mm, respectively, when compared with commercial antibiotic

Scheme 1 Synthesis of title compound 4 via oxidation of 3. Ar: Phenyl (a); 4-methylphenyl (b); 4-methoxyphenyl (c); 4-fluorophenyl (d); 4-chlorophenyl (e); 4-bromophenyl (f); 4-nitrophenyl (g).

Scheme 2 Mechanism for the oxidation of 3 to 4.

Prakash et al. Organic and Medicinal Chemistry Letters 2011, 1:1 http://www.orgmedchemlett.com/content/1/1/1

Page 3 of 9

Table 1 In vitro antibacterial activity of compounds 3 and 4

Compounds 3a 3b 3c 3d 3e 3f 3g 4a 4b 4c 4d 4e 4f 4g Ciprofloxacin Diameter of growth of inhibition zone (mm)a Staphylococcus aureus 17.3 16.6 17.3 18.6 16.3 16.3 17.0 22.6 21.3 20.3 17.0 15.5 21.6 18.6 27.6 Bacillus subtilis 19.3 17.3 18.6 19.3 17.6 17.6 18.3 24.6 24.6 21.6 18.6 16.0 22.6 19.3 26.3 Escherichia coli 13.3 13.0 14.3 14.2 13.8 19.6 18.0 16.3 15.6 17.3 14.6 25.0 Pseudomonas aeruginosa 15.6 13.2 12.8 15.0 13.5 14.3 17.3 16.6 15.3 13.6 16.2 15.6 15.3 25.3

-, no activity a Values including diameter of the well (8 mm), are means of three replicates

ciprofloxacin, which showed maximum zone of inhibition of 27.6 and 26.3 mm against Gram-positive bacteria, S. aureus and B. subtilis, respectively. Compounds 4a, 4b, 4c and 4f were found to be the most effective against both Gram-positive bacteria showing maximum zone of inhibition ranging between 20.3 and 22.6 mm. Rest of compounds showed fair activity against Grampositive bacterial strains (Table 1, Figure 1). All the synthesized compounds showed fair activity against both Gram-negative bacterial strains. In the whole series, the MIC value of various synthesized compounds (3 and 4) ranges between 16 and 256 g/mL against Gram-positive and Gram-negative bacteria (Table 2, Figure 2). Out of compounds 3a-3g, compound 3d was found to be the most effective against both Gram-positive bacteria having the lowest MIC value 64 g/mL when compared

with commercial antibiotic ciprofloxacin, which showed MIC value 5 g/mL for both Gram-positive bacteria. Out of compounds 4a-4g, compounds 4a, 4b, 4c and 4f possessed good antibacterial activity against B. subtilis with MIC of 16, 16, 32 and 32 g/mL, respectively (Table 2, Figure 2).
In vitro antifungal activity

All the newly synthesized compounds (3 and 4) were also tested in vitro for their antifungal activity against two fungi, namely Aspergillus niger and Aspergillus flavus. Standard antibiotic fluconazole was used for comparison with antifungal activity shown by compounds 3a-g and 4a-g. A careful analysis of percentage mycelial growth inhibition revealed that compounds 3a, 3b, 3c and 3d exhibit good antifungal activity against both A. flavus and A. niger. Out of compounds 4a-g,

Figure 1 Comparison of diameter of growth of inhibition of compounds (3 and 4) and standard drug.

Prakash et al. Organic and Medicinal Chemistry Letters 2011, 1:1 http://www.orgmedchemlett.com/content/1/1/1

Page 4 of 9

Table 2 Minimum inhibitory concentration (g/mL) of compounds 3 and 4

Compounds 3a 3b 3c 3d 3e 3f 3g 4a 4b 4c 4d 4e 4f 4g Ciprofloxacin
NT, not tested.

Minimum inhibitory concentration (g/mL) Staphylococcus aureus 128 128 128 64 128 128 128 32 32 64 128 128 32 64 5 Bacillus subtilis 64 128 64 64 128 128 64 16 16 32 64 128 32 64 5 Escherichia coli 256 NT 256 256 256 NT 256 64 64 128 128 256 64 256 5 Pseudomonas aeruginosa 128 256 256 256 NT 256 256 64 128 128 256 128 128 256 5

compounds 4a, 4b, 4c and 4f showed excellent activity against both antifungal strains as shown in Table 3 and Figure 3. It indicates that fused triazoles 4a-g containing electron-releasing substituents (4a, 4b and 4c) at para position of aryl ring of pyrazole moiety are more antifungal than triazoles having electron-withdrawing groups (4d, 4e, 4f and 4g) at the same position. A careful analysis of MIC data revealed some interesting results (Table 2) which are as follows (i) Compounds 3a-g have shown marginal activity, but after oxidative cyclization of these compounds, it was found that antibacterial activity has been increased.

(ii) Inhibitory data of compounds 4a-g suggested that the replacement of para proton of aryl ring of pyrazole moiety (at 3-position) in 3-(3-aryl-1-phenyl1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-a]pyridines (4a-g) with electron-releasing groups the antibacterial activity increases while replacing the same aryl proton with electron-withdrawing group the antibacterial activity decreases. In compound 4b, the proton at para position of aryl ring of pyrazole moiety is replaced with methyl group, and in compound 4c, the proton at para position of aryl ring of pyrazole moiety is replaced with methoxy group, both of these compounds exhibited significant level of antibacterial activity. Compounds 4d, 4e, 4f (containing halogen at para position of aryl ring of pyrazole

Figure 2 Comparison of MIC of compounds (3 and 4) and standard drug.

Prakash et al. Organic and Medicinal Chemistry Letters 2011, 1:1 http://www.orgmedchemlett.com/content/1/1/1

Page 5 of 9

Table 3 In vitro antifungal activity of compounds 3 and 4

Compounds 3a 3b 3c 3d 3e 3f 3g 4a 4b 4c 4d 4e 4f 4g Fluconazole Mycelial growth of inhibition (%) Aspergillus niger 52.5 51.1 52.5 50.0 44.4 45.5 47.7 66.5 62.3 60.5 52.5 48.8 69.5 51.1 81.1 Aspergillus flavus 51.1 50.6 51.1 49.8 45.5 44.4 45.5 58.8 62.5 60.6 51.1 50.0 68.5 50.0 77.7

3-(3-aryl-1-phenyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-a] pyridines (4a-g) with electron-releasing groups the antibacterial activity increased while as the para proton of aryl ring of pyrazole moiety is replaced with electron-withdrawing group, the antibacterial activity decreases. Triazoles 4 having electron-releasing substituents at para position of aryl ring of pyrazole moiety (4a, 4b and 4c) are more antifungal than triazoles containing electron-withdrawing groups (4a, 4e, 4f and 4g) at the same position.

Experimental Melting points were taken on slides in an electrical apparatus Labindia visual melting range apparatus and are uncorrected. The IR spectra were obtained with a Buck Scientific IR M-500 spectrophotometer. The 1 H NMR spectra were recorded on a Bruker (300 MHz) spectrometer using tetramethylsilane as an internal standard. All the compounds gave satisfactory analytical results (within 0.4% of the theoretical values). The starting material 4-formylpyrazoles 2 were prepared by the literature method [32].
2-((3-Aryl-1-phenyl-1H-pyrazol-4-yl)methylene)-1-(pyridin2-yl)hydrazines (3)

moiety) and 4g (containing nitro group at para position of aryl ring of pyrazole moiety) have shown marginal activities against all four bacteria.

Conclusions We have described in this study an efficient and convenient synthesis of some new 3-(3-aryl-1-phenyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-a]pyridines (4a-g) via the oxidative cyclization of hydrazones 3, thereby emphasizing the increasing utility of hypervalent iodine(III)mediated methods. The antibacterial and antifungal activities of newly synthesized compounds 3 and 4 have proven them potent antibacterial and antifungal agents. Compounds 3a-3g have shown marginal activity. Inhibitory data of compounds 4a-4g suggested that by the replacement of para proton of aryl ring of pyrazole moiety in

General procedure: To the ethanolic solution of 2-pyridylhydrazine (1, 0.01 mol) was added appropriate 4-formylpyrazole (2, 0.01 mol), and the solution was refluxed for 4-5 min. The solvent was evaporated in vacuo to half its volume and cooled to room temperature. The solid obtained was filtered and washed with ethanol.
2-((1,3-Diphenyl-1H-pyrazol-4-yl)methylene)-1-(pyridin-2yl)hydrazine (3a)

Yield 96%, Mp 210C, IR (max, KBr): 3190 cm-1 (-NH str.); 1 H NMR (DMSO-d6 , 300 MHz): 6.71-6.74 (m, 1H), 7.18-7.21 (m, 1H, J = 9 Hz), 7.34-7.39 (m, 1H), 7.47-7.63 (m, 6H), 7.74-7.77 (m, 2H), 7.98-8.01 (m, 2H), 8.07-8.09 (d, 1H, J = 6 Hz), 8.17(s, 1H, N=CH), 8.93(s,

Mycelial growth of inhibition (%)

Aspergillus niger 90 80 70 60 50 40 30 20 10 0

Aspergillus flavus

Compounds/Antifungal drug

Figure 3 Comparison of antifungal activity of compounds (3 and 4) and standard drug.

4g Fl uc on az ol e

3c

4c

3b

3a

3e

3f

3d

4a

3g

4b

4d

4e

4f

Prakash et al. Organic and Medicinal Chemistry Letters 2011, 1:1 http://www.orgmedchemlett.com/content/1/1/1

Page 6 of 9

1H), 10.66 (bs, 1H, exchangeable with D2O); Anal. Calculated for C21H17N5: C 74.32, H 5.05, N 20.63; Found: C 74.35, H 5.04, N 20.64; ESI-MS m/z: 340.06 [M + 1]+.
2-((1-Phenyl-3-p-tolyl-1H-pyrazol-4-yl)methylene)-1(pyridin-2-yl)hydrazine (3b)

Yield 94%, Mp 206C, IR (max, KBr): 3194 cm-1 (-NH str.); 1 H NMR (DMSO-d 6 , 300 MHz): 2.40 (s, 3H), 6.73-6.74 (m, 1H), 7.20-7.23 (d, 1H, J = 9 Hz), 7.32-7.38 (m, 3H), 7.51-7.59 (d, 2H, J = 9 Hz), 7.62-7.65 (m, 3H), 7.98-8.09 (m, 3H), 8.15 (s, 1H), 8.93(s, 1H, N=CH), 10.69 (bs, 1H, exchangeable with D2O); Anal. Calculated for C22H19N5: C 74.77, H 5.42, N 19.82; Found: C 74.79, H 5.45, N 19.79; ESI-MS m/z: 354.12 [M + 1]+
2-((3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl) methylene)-1-(pyridin-2-yl)hydrazine (3c)

1H), 7.12-7.14 (m, 1H), 7.35-7.38 (m, 1H), 7.50-7.59 (m, 3H), 7.71 (m, 4H), 7.95-7.98 (d, 2H, J = 8.1 Hz), 8.068.07 (d, 1H, J = 4.5 Hz), 8.12 (s, 1H, N=CH), 8.93(s, 1H), 10.67 (bs, 1H, exchangeable with D2O); Anal. Calculated for C 21 H 16 BrN 5 : C 60.30, H 3.86, N 16.74; Found: C 60.28, H 3.83, N 16.77; ESI-MS m/z: 385.12 ([M + 1]+, 387.02 [M + 3]+.
2-((3-(4-Nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)1-(pyridin-2-yl)hydrazine (3g)

Yield 89%, Mp 208C, IR (max, KBr): 3189 cm-1 (-NH str.); 1 H NMR (DMSO-d 6 , 300 MHz): 3.84(s, 3H), 6.70-6.74 (m, 1H), 7.07-7.10 (d, 2H, J = 9 Hz), 7.19-7.22 (m, 1H), 7.45-7.38 (m, 1H), 7.51-7.64 (m, 3H), 7.67-7.70 (d, 2H, J = 9 Hz), 7.97-8.00 (m, 2H), 8.07-8.09 (d, 1H, J = 6 Hz), 8.14 (s, 1H, N=CH), 8.91(s, 1H), 10.67 (bs, 1H, exchangeable with D 2 O); Anal. Calculated for C22H19N5O: C 71.53, H 5.18, N 18.96; Found: C 71.53, H 5.20, N 18.92; ESI-MS m/z: 370.08 [M + 1]+.
2-((3-(4-Fluorophenyl)-1-phenyl-1H-pyrazol-4-yl) methylene)-1-(pyridin-2-yl)hydrazine (3d)

Yield 92%, Mp 234C, IR (max, in KBr): 3195 cm-1 (-NH str.) 1335, 1504 (-NO 2 ); 1 H NMR (DMSO-d 6 , 300 MHz): 6.72-6.76 (m, 1H), 7.12-7.15 (m, 1H), 7.38-7.42 (m, 1H), 7.54-7.63 (m, 3H), 8.00-8.03 (m, 2H, J = 9 Hz), 8.08-8.11 (m, 3H), 8.20 (s, 1H, N=CH), 8.36-8.39 (d, 2H, J = 9 Hz), 9.01 (s, 1H), 10.76 (bs, 1H, exchangeable with D 2 O); Anal. Calculated for C 21 H 16 N 6 O 2 : C 65.62, H 4.20, N 21.86; Found: C 65.64, H 4.26, N 21.85; ESI-MS m/z: 267.06 [M + 1]+.
Synthesis of 3-(3-aryl-1-phenyl-1H-pyrazol-4-yl)-[1,2,4] triazolo[4,3-a]pyridines (4)

Yield 90%, Mp 224C, IR (max, KBr): 3198 cm-1 (-NH str.); 1 H NMR (DMSO-d6, 300 MHz): 6.71-6.75 (m, 1H), 7.147.17 (m, 1H), 7.33-7.39 (m, 3H), 7.52-7.63 (m, 3H), 7.797.84 (d, 2H, J = 9 Hz), 7.97-8.00 (d, 2H, J = 9 Hz), 8.088.09 (d, 1H, J = 3 Hz), 8.15 (s, 1H, N=CH), 8.93 (s, 1H), 10.66 (bs, 1H, exchangeable with D2O); Anal. Calculated for C21H16FN5: C 70.58, H 4.51, N 19.60; Found: C 70.59, H 4.53, N 19.58; ESI-MS m/z: 358.10 [M + 1]+.
2-((3-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-4-yl) methylene)-1-(pyridin-2-yl)hydrazine (3e)

General procedure: To a suspension/solution of 3 (0.010 mol) in dichloromethane (25 mL), IBD (0.011 mol) was added in small portions, and the reaction mixture was stirred overnight. Then, the solvent was evaporated on water bath. To the resulting residue was added ethanol (5-10 mL), and the mixture was warmed to obtain a clear solution. On cooling at room temperature, solid separated out was filtered and washed with cold alcohol to give pure fused 1,2,4-triazole derivatives 4a-g.
(1,3-Diphenyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-a]pyridine (4a)

Yield 90%, Mp 232C, IR (max, KBr): 3200 cm-1 (-NH str.); 1 H NMR (DMSO-d6 , 300 MHz): 6.70-6.74 (m, 1H), 7.13-7.15 (m, 1H), 7.34-7.39 (m, 1H), 7.52-7.62 (m, 5H), 7.79-7.82 (d, 2H, J = 9 Hz), 7.96-7.99 (d, 2H, J = 9 Hz), 8.07-8.09 (d, 1H, J = 6 Hz), 8.15 (s, 1H, N=CH), 8.91 (s, 1H), 10.64 (bs, 1H, exchangeable with D 2 O); Anal. Calculated for C 21 H 16 ClN 5 : C 67.47, H 4.31, N 18.73; Found: C 67.41, H 4.30, N 18.75; ESI-MS m/z: 374.10 [M + 1]+, 376.10 [M + 3]+.
2-((3-(4-Bromophenyl)-1-phenyl-1H-pyrazol-4-yl) methylene)-1-(pyridin-2-yl)hydrazine (3f)

Yield 90%, Mp 162C, IR ( max , KBr): transparent in the region of -NH str.; 1 H NMR (DMSO-d 6 , 300 MHz): 6.89-6.94 (m, 1H), 7.33-7.35 (m, 3H), 7.387.45 (m, 2H), 7.54-7.63 (m, 4H), 7.84-7.87 (s, 1H), 7.99-8.05 (m, 2H), 8.15-8.17 (d, 1H, J = 6 Hz), 9.16 (s, 1H); Anal. Calculated for C21 H 15 N5 : C 74.76, H 4.48, N 20.76; Found: C 74.77, H 4.45, N 20.73; ESI-MS m/ z: 338.09 [M + 1]+.
3-(1-Phenyl-3-p-tolyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-a] pyridine (4b)

Yield 91%, Mp 236C, IR (max, in KBr): 3203 cm-1 (-NH str.); 1 H NMR (DMSO-d6 , 300 MHz): 6.69-6.73 (m,

Yield 88%, Mp 210C, IR (max, KBr): transparent in the region of -NH str.; 1H NMR (DMSO-d6, 300 MHz): 2.28 (s, 3H) 6.89-6.93 (m, 1H), 7.13-7.16 (m, 2H), 7.387.44 (m, 4H), 7.57-7.62 (m, 2H), 7.80-7.84 (m, 1H), 8.01-8.03 (d, 2H, J = 6 Hz), 8.12-8.14 (d, 1H, J = 6 Hz), 9.13 (s, 1H); Anal. Calculated for C22H17N5: C 75.19, H 4.88, N 19.93; Found: C 75.18, H 4.86, N 19.96; ESI-MS m/z: 352.11 [M + 1]+.

Prakash et al. Organic and Medicinal Chemistry Letters 2011, 1:1 http://www.orgmedchemlett.com/content/1/1/1

Page 7 of 9

3-(3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-[1,2,4] triazolo[4,3-a]pyridine (4c)

Biological assay
Test microorganisms

Yield 82%, Mp 164C, IR (max, KBr): transparent in the region of -NH str.; 1H NMR (DMSO-d6, 300 MHz): 3.73 (s, 3H) 6.89-6.94 (m, 3H), 7.36-7.40 (m, 2H), 7.477.49 (m, 2H), 7.56-7.61 (m, 2H), 7.80-7.87 (m, 1H), 7.95-8.02 (m, 2H), 8.14-8.16 (m, 1H), 9.12 (s, 1H); Anal. Calculated for C 22 H 17 N 5 O: C 71.92, H 4.66, N 19.06; Found: C 71.91, H 4.64, N 19.07; ESI-MS m/z: 368.06 [M + 1]+.
3-(3-(4-Fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-[1,2,4] triazolo[4,3-a]pyridine (4d)

Yield 85%, Mp 193C, IR (max, KBr): transparent in the region of -NH st.; 1 H NMR (DMSO-d 6 , 300 MHz): 6.92-6.97 (m, 1H), 7.17-7.23 (m, 2H), 7.39-7.44 (m, 2H), 7.57-7.68 (m, 4H), 7.85-7.88 (m, 1H), 8.02-8.05 (d, 2H, J = 9 Hz), 8.24-8.26 (d, 1H, J = 6 Hz), 9.19 (s, 1H); Anal. Calculated for C 21 H 14 FN 5 : C 70.98, H 3.97, N 19.71; Found: C 70.98, H 3.99, N 19.69; ESI-MS m/z: 356.08 [M + 1]+.
3-(3-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-[1,2,4] triazolo[4,3-a]pyridine (4e)

Total six microbial strains were selected on the basis of their clinical importance in causing diseases in humans. Two Gram-positive bacteria (Staphylococcus aureus MTCC 96 and Bacillus subtilis MTCC 121); two Gramnegative bacteria (Escherichia coli MTCC 1652 and Pseudomonas aeruginosa MTCC 741) and two fungi, Aspergillus niger and A. flavus, the ear pathogens isolated from the patients of Kurukshetra, were used in the present study for the evaluation of antimicrobial activity of the compounds [33]. All the cultures were procured from Microbial Type Culture Collection (MTCC), IMTECH, Chandigarh. The bacteria were subcultured on nutrient agar, whereas fungi on Sabouraud dextrose.
In vitro antibacterial activity

Yield 89%, Mp 133C, IR (max, KBr): transparent in the region of -NH str.; 1H NMR (DMSO-d6, 300 MHz): 6.93-6.98 (m, 1H), 7.41-7.45 (m, 4H), 7.57-7.64 (m, 4H), 7.85-7.88 (m, 1H), 8.02-8.04 (d, 2H, J = 6 Hz), 8.25-8.27 (d, 1H, J = 6 Hz), 9.19 (s, 1H); Anal. Calculated for C21H14ClN5: C 67.83, H 3.80, N 18.84; Found: C 67.81, H 3.84, N 18.80; ESI-MS m/z: 372.01 [M + 1]+, 374.06 [M + 3]+.
3-(3-(4-Bromophenyl)-1-phenyl-1H-pyrazol-4-yl)-[1,2,4] triazolo[4,3-a]pyridine (4f)

Yield 84%, Mp 140C, IR (max, KBr): transparent in the region of -NH str.; 1H NMR (DMSO-d6, 300 MHz): 6.92-6.96 (m, 1H), 7.38-7.43 (m, 2H), 7.54-7.60 (m, 6H), 7.83-7.86 (m, 1H), 7.99-8.02 (d, 2H, J = 8.1 Hz), 8.238.26 (d, 1H, J = 6.9 Hz), 9.17 (s, 1H); Anal. Calculated for C 21 H 14 BrN 5 : C 60.59, H 3.39, N 16.82; Found: C 60.56, H 3.37, N 16.81; ESI-MS m/z: 416.04 [M + 1]+, 418.06 [M + 3]+.
3-(3-(4-Nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)-[1,2,4] triazolo[4,3-a]pyridine (4g)

The antibacterial activity of chemical compounds was evaluated by the agar well diffusion method. All the cultures were adjusted to 0.5 McFarland standards, which is visually comparable to a microbial suspension of approximately 1.5 108 cfu/mL. 20 mL of Mueller Hinton agar medium was poured into each Petri plate, and the agar plates were swabbed with 100 L inocula of each test bacterium and kept for 15 min for adsorption. Using sterile cork borer of 8 mm diameter, wells were bored into the seeded agar plates, and these were loaded with a 100 L volume with concentration of 2.0 mg/mL of each compound reconstituted in the dimethylsulphoxide (DMSO). All the plates were incubated at 37C for 24 h. Antibacterial activity of each compound was evaluated by measuring the zone of growth inhibition against the test organisms with zone reader (Hi Antibiotic zone scale). The DMSO was used as a negative control, whereas ciprofloxacin was used as a positive control. This procedure was performed in three replicate plates for each organism [34].

Yield 89%, Mp 242C, IR (max, KBr): transparent in the region of -NH str.; 1H NMR (DMSO-d6, 300 MHz): 6.82-6.86 (m, 1H), 7.34-7.41 (m, 1H), 7.44-7.46 (m, 1H), 7.55-7.60 (m, 3H), 7.83-7.87 (m, 3H), 7.92-7.94 (d, 2H, J = 6 Hz), 8.14-8.17 (d, 2H, J = 9 Hz), 8.71 (s, 1H); Anal. Calculated for C21H14N6O2: C 65.96, H 3.69, N 21.98; Found: C 65.97, H 3.67, N 21.94; ESI-MS m/z: 383.10 [M + 1]+.

Determination of minimum inhibitory concentration (MIC) The MIC is the lowest concentration of an antimicrobial compound that will inhibit the visible growth of a microorganism after overnight incubation. The MIC of all the synthesized compounds (3 and 4) against bacterial strains was tested through a modified agar well-diffusion method [35]. In this method, a twofold serial dilution of each compound was prepared by first reconstituting the compound in DMSO followed by dilution in sterile, distilled water to achieve a decreasing concentration range of 256-0.5 g/mL. A 100 L volume of each dilution was introduced into wells (in triplicate) in the agar plates already seeded with 100 L of standardized inoculum (106 cfu/mL) of the test microbial strain. All test plates were incubated aerobically at 37C for 24

Prakash et al. Organic and Medicinal Chemistry Letters 2011, 1:1 http://www.orgmedchemlett.com/content/1/1/1

Page 8 of 9

h and observed for the inhibition zones. The MIC, taken as the lowest concentration of the chemical compound that completely inhibited the growth of the microbe, showed by a clear zone of inhibition, was recorded for each test organism. Ciprofloxacin was used as positive control while DMSO as negative control.
In vitro antifungal activity

Additional file 12: 1H NMR spectra. (4e): 1H NMR of 3-(3-(4Chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-a]pyridine. Additional file 13: 1H NMR spectra. (4f): 1H NMR of 3-(3-(4Bromophenyl)-1-phenyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-a]pyridine. Additional file 14: 1H NMR spectra. (4g): 1H NMR of 3-(3-(4Nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-a]pyridine.

The antifungal activity of the newly synthesized compounds was evaluated by poison food technique. The moulds were grown on Sabouraud dextrose agar (SDA) at 25C for 7 days and used as inocula. 15 mL of molten SDA (45C) was poisoned by the addition of 100 L volume of each compound having concentration of 4.0 mg/mL, reconstituted in the DMSO, poured into a sterile Petri plate and allowed to solidify at room temperature. The solidified poisoned agar plates were inoculated at the centre with fungal plugs (8 mm diameter), obtained from the actively growing colony and incubated at 25C for 7 days. The DMSO was used as the negative control, whereas fluconazole was used as the positive control. The experiments were performed in triplicates. Diameter of the fungal colonies was measured and expressed as percent mycelial inhibition determined by applying the following formula [36]:

Abbreviations DMSO: dimethylsulphoxide; IBD: iodobenzene diacetate; MIC: minimum inhibitory concentration; MTCC: microbial type culture collection; SDA: sabouraud dextrose agar. Acknowledgements The authors are thankful to the CSIR and the UGC, New Delhi for the award of Junior Research Fellowship to Khalid Hussain and Deepak K. Aneja. Thanks are also due to the RSIC, CDRI Lucknow, India, for providing mass and elemental analyses. Author details 1 Institute of Pharmaceutical Sciences, Kurukshetra University, Kurukshetra 136119, Haryana, India 2Department of Chemistry, Kurukshetra University, Kurukshetra 136119, Haryana, India 3Department of Microbiology, Kurukshetra University, Kurukshetra 136119, Haryana, India Competing interests The authors declare that they have no competing interests. Received: 23 March 11 Accepted: 18 July 11 Published: 18 July 11 References 1. Parmar SS, Gupta AK, Singh HH, Gupta TK (1972) Benzimidazolyl-1,2,4-(H)triazoles as central nervous system depressants. J Med Chem 15:9991000 2. Hwang LC, Tu CH, Wang JH, Lee GH (2006) Synthesis and molecular structure of 6-amino-3-benzylmercapto-1,2,4-triazolo[3,4-f][1,2,4]triazin-8(7H)one. Molecules 11:169176 3. Hiremath SP, Ullagaddi A, Shivaramayya K, Purohit MG (1999) Amino acid derivatives, VI [1]: synthesis, antiviral, and antimicrobial evaluation of amino acid esters bearing a 1,2,3-triazolo[4,5-d]pyrimidinedione side chain. Indian J Heterocycl Chem 3:145148 4. Zitouni GT, Kaplancikl ZA, zdemir A, Chevallet P, Kandilci HB, Gmsel B (2007) Studies on 1,2,4-triazole derivatives as potential anti-inflammatory agents. Arch Pharm Chem Life Sci 11:586590 5. Bower JD, Doyle FP (1957) The preparation of fused triazole systems. J Chem Soc 727732 6. Pollak A, Tiler M (1966) Synthesis of pyridazine derivativesV: formation of s-triazolo-(4,3-b)-pyridazines and bis-s-triazolo-(4,3-b,3,4-f)-pyridazines. Tetrahedron 22:20732079 7. Gibson MS (1963) HydrazonesIV: the bromination of benzylidene 2pyridylhydrazone. Tetrahedron 19:15871589 8. Moriarty RM, Vaid RK, Koser GF (1990) [Hydroxy(organosulfonyloxy)iodo] arenes in organic synthesis. Synlett 7:365383 9. Varvoglis A (1997) Hypervalent iodine in organic synthesis. Academic Press, London 10. Moriarty RM, Prakash O (1997) Synthesis of heterocyclic compounds using organohypervalent iodine reagents. Adv Heterocycl Chem 69:187 11. Chiai M, Akibia K (1999) Chemistry of hypervalent compounds. VCH Publishers, New York,chap 13: pp 59387 12. Moriarty RM, Prakash O (2001) Oxidation of phenolic compounds with organohypervalent iodine reagents. In: Overman LE, et al (eds) Organic reactions, vol 57. John Wiley & Sons Inc, pp 327415 13. Zhdankin VV, Stang PJ (2002) Recent developments in the chemistry of polyvalent iodine compounds. Chem Rev 102:25232584 14. Prakash O, Singh SP (1994) Iodobenzene diacetate and related hypervalent iodine reagents in the synthesis of heterocyclic compounds. Aldrichim Acta 27:1522 15. Varvoglis A (1997) Chemical transformations induced by hypervalent iodine reagents. Tetrahedron 53:11791255

dc, average diameter of fungal colony in negative control plates; dt average diameter of fungal colony in experimental plates.

Additional material
Additional file 1: 1H NMR spectra. (3a): 1H NMR of 2-((1,3-Diphenyl-1Hpyrazol-4-yl)methylene)-1-(pyridin-2-yl)hydrazine. Additional file 2: 1H NMR spectra. (4a): 1H NMR of (1,3-Diphenyl-1Hpyrazol-4-yl)-[1,2,4]triazolo[4,3-a]pyridine. Additional file 3: 1H NMR spectra. (3b): 1H NMR of 2-((1-Phenyl-3-ptolyl-1H-pyrazol-4-yl)methylene)-1-(pyridin-2-yl)hydrazine. Additional file 4: 1H NMR spectra. (3c): 1H NMR of 2-((3-(4Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-1-(pyridin-2-yl)hydrazine. Additional file 5: 1H NMR spectra. (3d): 1H NMR of 2-((3-(4Fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-1-(pyridin-2-yl)hydrazine. Additional file 6: 1H NMR spectra. (3e): 1H NMR of 2-((3-(4Chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-1-(pyridin-2-yl)hydrazine. Additional file 7: 1H NMR spectra. (3f): 1H NMR of 2-((3-(4Bromophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-1-(pyridin-2-yl)hydrazine. Additional file 8: 1H NMR spectra. (3g): 1H NMR of 2-((3-(4-Nitrophenyl)1-phenyl-1H-pyrazol-4-yl)methylene)-1-(pyridin-2-yl)hydrazine. Additional file 9: 1H NMR spectra. (4b): 1H NMR of 3-(1-Phenyl-3-p-tolyl1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-a]pyridine. Additional file 10: 1H NMR spectra. (4c): 1H NMR of 3-(3-(4Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-a]pyridine. Additional file 11: 1H NMR spectra. (4d): 1H NMR of 3-(3-(4Fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-a]pyridine.

Prakash et al. Organic and Medicinal Chemistry Letters 2011, 1:1 http://www.orgmedchemlett.com/content/1/1/1

Page 9 of 9

16. Wirth T, Chiai M, Zhdankin VV, Koser GF, Tohma H, Kita Y (2003) Hypervalent iodine chemistry-modern developments in organic synthesis. In Topics in Current Chemistry, vol 224.Springer, Berlin 17. Zhdankin VV (2009) Hypervalent iodine (III) reagents in organic synthesis. Arkivoc 1:162 18. Sadana AK, Mirza Y, Aneja KR, Prakash O (2003) Hypervalent iodine mediated synthesis of 1-aryl/hetryl-1,2,4-triazolo[4,3-a] pyridines and 1-aryl/ hetryl 5-methyl-1,2,4-triazolo[4,3-a]quinolines as antibacterial agents. Eur J Med Chem 38:533536 19. Prakash O, Bhardwaj V, Kumar R, Tyagi P, Aneja KR (2004) Organoiodine (III) mediated synthesis of 3-aryl/hetryl-5,7-dimethyl-1,2,4-triazolo[4,3-a] pyrimidines as antibacterial agents. Eur J Med Chem 39:10731077 20. Prakash O, Kumar R, Kumar R, Tyagi P, Kuhad RC (2007) Organoiodine(III) mediated synthesis of 3,9-diaryl- and 3,9-difuryl-bis-1,2,4-triazolo[4,3-a][4,3-c] pyrimidines as antibacterial agents. Eur J Med Chem 42:868872 21. Prakash O, Kumar R, Sharma D, Naithani R, Kumar R (2006) Organoiodine(III)mediated efficient synthesis of new 3,9-diaryl-bis-1,2,4-triazolo[4,3-a][4,3-c] pyrimidines. Heteroatom Chem 17:653655 22. Kumar R, Nair RR, Dhiman SS, Sharma J, Prakash O (2009) Organoiodine (III)mediated synthesis of 3-aryl/heteroaryl-5,7-dimethyl-1,2,4-triazolo[4,3-c] pyrimidines as antibacterial agents. Eur J Med Chem 44:22602264 23. Ursic U, Bevk D, Pirc S, Pezdirc L, Stanovnik B, Svete J (2006) Enaminonebased synthesis of (S)-3-(pyrazolyl)alanines from L-aspartic acid. Synthesis 14:23762384 24. Bebernitz GR, Argentieri G, Battle B, Brennan C, Balkan B, Bryan F, Burkey BF, Eckhardt M, Gao J, Kapa P, Strohschein RJ, Schuster HF, Wilson M, Xu DD (2001) The effect of 1,3-diaryl-[1H]-pyrazole-4-acetamides on glucose utilization in ob/ob Mice. J Med Chem 44:26012611 25. Kameyama T, Ukai M, Nabeshima T (1978) Inhibitory effect of difenamizole on morphine- induced straub tail reaction with special reference to monoaminergic agents. Chem Pharm Bull 26:32653270 26. Escriv E, Lozano JG, Lillo JM, Nuez H, Carri JS, Soto L, Carrasco R, Cano J (2003) Synthesis, crystal structure, magnetic properties, and theoretical studies of [{Cu(mepirizole)Br}2(-OH)(-pz)] (mepirizole = 4-methoxy-2-(5methoxy-3-methyl-1H-pyrazol-1-yl)-6-methylpyrimidine; pz = pyrazolate), a novel -pyrazolato--hydroxo-dibridged copper(II) complex. Inorg Chem 42:83288336 27. McQuay HJ, Moore RA (2005) NSAIDS and coxibs: clinical use. In: McMahon S, Koltzenburg M (eds) Wall and Melzacks textbook of pain. Churchill Livingstone, Edinburgh pp 471480 28. Jayashankaran J, Rathna Durga RSM, Raghunathan R (2006) An efficient synthesis of thiopyrano[5,6-c]coumarin/[6,5-c]chromones through intramolecular domino Knoevenagel hetero Diels-Alder reactions. Tetrahedron Lett 47:22652270 29. Bauer VJ, Dalalian HP, Fanshawe SR, Safir SR, Tocus EC, Benedict A (1968) 4[3(5)-Pyrazolyl]pyridinium salts. A new class of hypoglycaemic agents. J Med Chem 11:981984 30. Genin MJ, Biles C, Keiser BJ, Poppe SM, Swaney SM, Tarpley WG, Yagi Y, Romero DL (2000) Novel 1,5-diphenylpyrazole nonnucleoside HIV-1 reverse transcriptase inhibitors with enhanced activity versus the delavirdineresistant P236L mutant: lead identification and SAR of 3- and 4-substituted derivatives. J Med Chem 43:10341040 31. Okimoto M, Chiba T (1990) Electrochemical oxidation of ketone acylhydrazones and their hydrogen cyanide adducts in sodium cyanidemethanol. Transformation of ketones to nitriles. J Org Chem 55:10701076 32. Kira MA, Abdel-Rahman MO, Gadalla KZ (1969) The vilsmeier-haack reactionIII cyclization of hydrazones to pyrazoles. Tetrahedron Lett 10:109110 33. Aneja KR, Sharma C, Joshi R (2010) Fungal infection of ear: a common problem in north eastern part of Haryana. Inter J Otorhinolaryngol 74:604607 34. Ahmad I, Beg AJ (2001) Antimicrobial and phytochemical studies on 45 Indian medicinal plants against multidrug resistant human pathogens. J Ethnopharmacol 74:113123 35. Okeke MI, Iroegbu CU, Eze EN, Okoli AS, Esimone CO (2001) Evaluation of extracts of the roots of Landolphia owerrience for antibacterial activity. J Ethnopharmacol 78:119127 36. Al-Burtamani SKS, Fatope MO, Marwah RG, Onifade AK, Al-Saidi SH (2005) Chemical composition, antibacterial and antifungal activities of the essential oil of Haplophyllum tuberculatum from Oman. J Ethnopharmacol 96:107112

doi:10.1186/2191-2858-1-1 Cite this article as: Prakash et al.: A facile iodine(III)-mediated synthesis of 3-(3-aryl-1-phenyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-a]pyridines via oxidation of 2-((3-aryl-1-phenyl-1H-pyrazol-4-yl)methylene)-1-(pyridin-2yl)hydrazines and their antimicrobial evaluations. Organic and Medicinal Chemistry Letters 2011 1:1.

Submit your manuscript to a journal and benet from:

7 Convenient online submission 7 Rigorous peer review 7 Immediate publication on acceptance 7 Open access: articles freely available online 7 High visibility within the eld 7 Retaining the copyright to your article

Submit your next manuscript at 7 springeropen.com